This invention relates to influenza virus immunogenic compositions and methods of producing such compositions. More specifically, this invention relates to influenza virus immunogenic compositions having discreet, specifically engineered mutations in the native PB2 polymerase RNA sequence of influenza resulting in the deletion of, and/or substitution of, at least one of the native tryptophan amino acid residues in the PB2 protein.
Influenza is an enveloped, single-stranded, negative-sense RNA virus that causes serious respiratory ailments throughout the world. It is the only member of the Orthomyxoviridae family and has been subgrouped into three types, A, B and C.
Influenza virions consist of an internal ribonucleoprotein core containing the single-stranded RNA genome and an outer lipoprotein envelope lined inside by a matrix (hereinafter xe2x80x9cM1xe2x80x9d) protein. The segmented genome of influenza A consists of eight molecules of linear, negative polarity, single-stranded RNA sequences that encode ten polypeptides. Segment 1 is 2341 nucleotides in length and encodes PB2, a 759 amino acid polypeptide which is one of the three proteins which comprise the RNA-dependent RNA polymerase complex. The remaining two polymerase proteins, PB1, a 757 amino acid polypeptide, and PA, a 716 amino acid polypeptide, are encoded by a 2341 nucleotide sequence and a 2233 nucleotide sequence (segments 2 and 3), respectively. Segment 4 of the genome consists of a 1778 nucleotide sequence encoding a 566 amino acid hemagglutin (HA) surface glycoprotein which projects from the lipoprotein envelope and mediates attachment to and entry into cells. Segment 5 consists of 1565 nucleotides encoding a 498 amino acid nucleoprotein (NP) protein that forms the nucleocapsid. Segment 6 consists of a 1413 nucleotide sequence encoding a 454 amino acid neuraminidase (NA) envelope glycoprotein. Segment 7 consists of a 1027 nucleotide sequence encoding a 252 amino acid M1 protein, and a 96 amino acid M2 protein, which is translated from a spliced variant of the M RNA. Segment 8 consists of a 890 nucleotide sequence encoding two nonstructural proteins, NS 1 and NS2, composed of 230 and 121 amino acids respectively, whose function is not well defined. NS2 is translated from a spliced variant of the NS RNA.
The segmented genome of influenza B consists of eight molecules of linear, negative polarity, single-stranded RNA sequences that encode eleven polypeptides. Segment 2 is 2396 nucleotides in length and encodes PB2, a 770 amino acid polypeptide which is one of the three RNA-dependent RNA polymerase proteins. The remaining two influenza B polymerase proteins, PB 1, a 752 amino acid polypeptide, and PA, a 725 amino acid polypeptide, are encoded by a 2386 nucleotide sequence and a 2304 nucleotide sequence (segments 1 and 3), respectively. Segment 4 of the genome consists of a 1882 nucleotide sequence encoding a 584 amino acid HA surface glycoprotein which projects from the lipoprotein envelope and mediates attachment to cells and membrane fusion. Segment 5 consists of 1839-1841 nucleotides encoding a 560 amino acid NP protein that forms the nucleocapsid. Segment 6 consists of a 1454 nucleotide sequence encoding a 466 amino acid NA envelope glycoprotein and a 100 amino acid NB protein, a nonstructural protein whose function is unknown. Segment 7 consists of a 1191 nucleotide sequence encoding a 248 amino acid M1 protein and a 195 amino acid BM2 protein which is translated from a separate reading frame. Segment 8 consists of a 1096 nucleotide sequence encoding nonstructural proteins NS 1 and NS2, composed of 281 and 122 amino acids respectively, whose functions are not well defined. NS2 is translated from a spliced variant of the NS RNA.
The segmented genome of influenza C consists of seven molecules of linear, negative polarity, single-stranded RNA sequences that encode eight polypeptides. Segment 1 is 2365 nucleotides in length and encodes PB2, a 774 amino acid polypeptide which is one of the three RNA-dependent RNA polymerase proteins. The remaining two polymerase proteins, PB 1, a 754 amino acid polypeptide, and PA, a 709 amino acid polypeptide, are encoded by a 2363 nucleotide sequence and a 2183 nucleotide sequence (segments 2 and 3), respectively. Segment 4 of the genome consists of a 2074 nucleotide sequence encoding a 655 amino acid hemagglutinin-esterase surface glycoprotein which projects from the lipoprotein envelope and mediates attachment to cells, fusion, and has receptor-destroying activities. Segment 5 consists of a 1809 nucleotide sequence encoding a 565 amino acid NP protein that forms the nucleocapsid. Segment 6 consists of a 1180 nucleotide sequence encoding a 374 amino acid matrix (M) protein. Segment 7 consists of a 934 nucleotide sequence encoding a 286 amino acid NS 1 protein, and a 122 amino acid NS2 protein, which is translated from a spliced variant of the NS RNA.
To infect a cell influenza HA protein adsorbs to sialyloligosaccharide molecules in cell membrane glycoproteins and glycolipids. Following endocytosis of the virion, a conformational change in the HA molecule occurs within the cellular endosome that facilitates membrane fusion and triggers uncoating. The nucleocapsid migrates to the nucleus where viral mRNA is transcribed as the essential initial event in infection. Transcription and replication of influenza RNA take place in the nucleus of infected cells and assembly into virions occurs by budding out of or through the plasma membrane. Viruses can reassort genes during mixed infections.
Replication of influenza virus RNAs is dependent on four viral gene products: PB1, PB2, PA, and NP. The three polymerase proteins, PB1, PB2, and PA, form a trimolecular complex in the nuclei of infected cells. Some specific functions have been ascribed to the individual polypeptides. PB1 appears to be primarily involved in the enzymatic polymerization process, i.e. the elongation step. It shares regions of amino acid sequence similarity with other RNA-dependent RNA polymerase proteins. The precise function of PA is unknown. The PB2 protein binds to the 5xe2x80x2-terminal cap structure present on host cell mRNAs; the mRNAs are then cleaved, producing a capped 9 to 15-mer oligoribonucleotide which serves as a primer for transcription of influenza mRNAs. See Plotch, Cell 23: 847-58 (1981). Thus, it is suspected that PB2 has cap-binding and endonuclease activities. While it is thought that PB2 is not absolutely required for replication of viral RNA, mRNAs transcribed from viral template in cells expressing only PB1, PA, and NP are uncapped and thus cannot be translated. See Nakagawa, J Virol 69:728-33 (1995). Transcripts terminate at sites 15-22 bases from the ends of their templates, where oligo(U) sequences act as signals for the template-independent addition of poly(A) tracts. At a later stage of infection, instead of making mRNAs, the polymerase proteins PB1, PB2 and PA are used to make new viral RNA genomes. The polymerase complex transcribes cRNA, which then serves as template for production of more vRNA. The plus-stranded cRNA copies differ from the plus-stranded mRNA transcripts by lacking capped and methylated 5xe2x80x2-termini. Also, they are not truncated or polyadenylated at the 3xe2x80x2 termini. Thus, the cRNAs are coterminal with their negative strand templates and contain all the genetic information in each genomic segment in the complementary form.
The negative strand genomes (vRNAs) and antigenomes (cRNAs) are always encapsidated by viral nucleocapsid proteins; the only unencapsidated RNA species are virus mRNAs. Nucleocapsid assembly appears to take place in the nucleus. The virus matures by budding from the apical surface of the cell incorporating the M1 protein on the cytoplasmic side or inner surface of the budding envelope. The HA and NA glycoproteins are incorporated into the lipid envelope. In permissive cells, HA is post-translationally cleaved, but the two resulting chains remain associated by disulfide bonds.
Efforts to produce immunogenic compositions against influenza have taken two paths. Inactive vaccines, which cannot replicate in the host, can be either chemically inactivated whole virus or viral subunit proteins. Both inactivated and subunit virus vaccines are available for influenza. These vaccines contain the HA and NA surface proteins as antigens which give rise to the immune response upon administration to the host. For reasons which are incompletely understood, subunit vaccines have exhibited an efficacy of only 60% to 80% against influenza disease. Inactivated whole virus vaccines are administered intramuscularly and primarily stimulate a humoral immune response, whereas live attenuated vaccines also stimulate local mucosal immunity. The latter form of immunity is more effective since it is present in the upper respiratory tract where a wild-type virus would first be encountered. Also, inactivated vaccines typically have reduced ability to induce cytotoxic T cell responses, and can sometimes cause delayed hypersensitivity reactions. Guillain-Barre syndrome has been associated with the inactivated influenza A xe2x80x9cswine fluxe2x80x9d vaccine. See, Schonberger, Ann Neurol 9(supp):31-38(1981).
Live attenuated viruses can be employed in immunogenic compositions and are typically successful at inducing the required protective response in the host. Live attenuated influenza viruses are capable of limited replication in the host, thus stimulating a protective immune response, but without causing disease. In making such vaccine compositions, the HA and NA RNA sequences of the attenuated xe2x80x9cmaster donorxe2x80x9d virus are replaced with HA and NA RNA sequences from circulating influenza strains. Such viruses are termed reassortant viruses. Previously, such master donor viruses have been generated by multiple passage through an unnatural host such as embryonated chicken eggs, by reassortment of genes between human and avian influenza viruses, by successive passage through an unnatural host at increasingly lower temperatures, or by random mutagenesis via chemical methods and selection of conditional mutants. These methods can result in the loss of pathogenicity while retaining immunogenicity. However, the identity of the genetic mutations generated as described above are unknown a priori and when the mutant xe2x80x9cmaster donorxe2x80x9d virus is selected as a vaccine candidate. If such mutations are limited to one or two nucleotide changes, the virus composition could ultimately xe2x80x9crevertxe2x80x9d or back mutate in the host and thus regain its original pathogenic phenotype. However, one of these methods, successive passage at increasingly lower temperatures, has given rise to a virus (the xe2x80x9ccold-adaptedxe2x80x9d strain derived from A/Ann Arbor/6/60) with multiple mutations that has been shown to be genetically stable. See Murphy, Inf Dis In Clin Practice 2:174-81 (1993). This cold-adapted vaccine may be highly effective in children and younger adults but appears to be less immunogenic in the elderly population. See Powers, J Am Ger Soc 40:163-67(1992).
Temperature sensitive (ts) mutants of influenza, generated by chemical mutagenesis or identified by screening for spontaneous mutants have been described. Such mutants are unable to replicate in the lower respiratory tract of infected animals and often replicate in the upper respiratory tract to a lower level than wild-type virus. One of these mutants, ts1A2, was shown to have many of the desired characteristics of a live attenuated influenza vaccine. See Murphy and Chanock, Genetic Variation Among Influenza Viruses, pps 601-15, Nayak, D.ed, Academic Press, NY (1981) and Murphy, Phil Trans R Soc Lon B 288:401-15(1980). The ts1A2 strain was found to contain temperature sensitive lesions in both PB1 and PB2, and exhibited the desired level of attenuation, but was genetically unstable and reverted to a virulent state after replication in a seronegative young vaccinee. See Murphy, Ann NYAcad Sci 354:17-82 (1980) and Tolpin, Infection and Immunity 36:64-50 (1982). Other ts mutants of influenza are known, the nucleotide sequences of their PB2 genes and the locations of the ts lesions in those genes have been determined. See Lawson, Virology 191:506-10(1992).
An alternate method of creating a live attenuated virus is by employing the techniques of xe2x80x9creverse geneticsxe2x80x9d. See Enami, Proc Natl Acad Sci 87:3802-05(1990), Enami and Palese, J Virol 65:2711-13(1991) and Luytjes, Cell 59:1107-13 (1989). In this process, modified vRNA▾like transcripts are transcribed in vitro from cDNA constructs in the presence of purified NP, PB1, PB2, and PA proteins. The resulting synthetic RNP is then transfected into cells previously infected with an influenza helper virus. This helper virus usually has a conditional growth defect, such as host range restriction or temperature sensitivity, which allows the subsequent selection of transfectant viruses. For example, host-range helper viruses have been successfully used to rescue synthetic NA and PB2 genes. See Enami, supra, and Subbarao, J Virol 67:7223-28 (1993). Antibody selection can also be used to select transfectants in the case of the HA and NA genes. Using antibody selection techniques, the surface HA glycoprotein gene has been transfected and rescued into influenza A virus. See, Horimoto and Kawaoka, J Virol 68:3120-28 (1994) and Li, J Virol 66:399-404(1992). A modified HA gene has also been transfected and rescued into influenza B virus. See, Barclay and Palese, J Virol 69:1275-79 (1995). The M gene (see, Yasuda, J Virol 68:8141-46 (1994)), and the NP gene (see Li, Virus Res 37:153-61(1995), have also been rescued using the techniques of reverse genetics.
Given the possibility of using reverse genetics to engineer specific mutations into the genome of influenza, it should be possible to create a virus strain with mutations that are less likely to revert and thus exhibit the desired property of genetic stability. This may be accomplished by introducing new codons which would require more than one nucleotide within the codon to mutate in order to encode the wild-type amino acid, by mutating sites which are less likely to be suppressed extragenically, by introducing multiple, independently-acting mutations in one or more genes, or by a combination of these approaches.
Studies with eukaryotic cellular cap-binding proteins have been largely confined to the eukaryotic translation initiation factor, eIF-4E. This 24 kilodalton (kD) protein binds to the cap structures on mRNAs and enables translation initiation in concert with a bevy of other eIFs. See Sonenberg, Prog Nucleic Acid Res Mol Biol 35:173-207(1988). Although the amino acids of the eIF-4E protein that interact directly with the cap structure have not been identified, biophysical studies have suggested the involvement of tryptophan residues in the eIF-4E protein. See Ishida, Biochem and Biophys Res Comm 115:849-54(1983). Site directed mutagenesis of tryptophan residues in the eIF-4E protein of Saccharomyces cerevisiae followed by assays for cap-binding suggested that two of the eight tryptophan residues present in the protein, those at the amino and/or carboxyl termini, might play a role in cap-recognition, while mutagenesis of certain other tryptophan residues resulted in mutated protein that still exhibited efficient cap-binding but reduced cross-linking ability relative to the wild-type protein. See Altmann, J Biol Chem 263:17229-32(1988).
The PB2 polypeptide has been shown to have cap-binding activity by cross-linking studies to cap analogs. By comparing the amino acid sequence of PB2 with those of the human and yeast cap-binding proteins, it has been theorized that the cap-binding activity in PB2 is located in two regions of the polypeptide sequence: amino acids 552-565 and amino acids 633-650. See de la Luna, Virus Res 13:143-56(1989). It has been speculated that one PB2 mutant, apparently having an inserted amino acid at position 299, is suspected of affecting cap binding or cap-dependent endonuclease activity. See Perales, J Virol 70:1678-86(1995). These authors also speculate that certain surrounding amino acids, presumably at positions 236, 469 and 480, define a region involved in cap binding in PB2. Id. at 1685.
In contrast to prior studies, we have identified one region, spanning PB2 amino acid residues 537-575, as most likely to contain the cap-binding activity. This region contains four tryptophan residues, at amino acid positions 537, 552, 557, and 564 (counting from the N-terminal MET, as residue 1). Additionally, we have found that modification of one or more of these tryptophan residues in the native PB2 protein of influenza, by deletion, or by substitution or replacement with a non-native residue, alone or in combination with other known PB2 ts lesions, results in the exhibition of attenuation of virulence and temperature sensitivity in influenza virus.
Accordingly, in one aspect the invention comprises novel PB2 tryptophan variant polypeptide sequences and RNA sequences encoding PB2 tryptophan variant polypeptides, which, when incorporated into influenza viral master donor viruses, cause such viruses to exhibit an attenuated and temperature sensitive phenotype. The PB2 tryptophan variant polypeptides of this invention comprise variant or modified PB2 amino acid sequences in which at least one and up to four of the tryptophan residues of wild-type (i.e., native) influenza PB2 sequences believed to be involved in cap-binding are modified by deletion or by replacement or substitution with non-native amino acids. The PB2 tryptophan variant polypeptides of this invention comprise variant or modified PB2 sequences which, in addition, may contain one or more amino acid substitutions known to be responsible for temperature-sensitivity. A number of such xe2x80x9ctsxe2x80x9d mutants of human influenza A/Udorn/307/72 virus are known. A summary of the nucleotide and deduced amino acid sequence changes in the PB2 RNA sequences of certain of these ts mutants is disclosed in Lawson, Virus Res 191:506-10(1992) and in PCT Patent Publication WO 95/08634 published Mar. 30, 1995, which are herein incorporated by reference. Of particular interest are A/Udorn/307/72 mutants in which the native E at position 65 is replaced with G (using the accepted one-letter abbreviations for amino acids) and in which the native P at position 112 is replaced with S in PB2. Other ts mutations encompassed by this invention include that found in the PB2 gene of the cold adapted strain of human influenza A/AA/6/60, in which the native N is replaced with S at amino acid position 265.
The invention also comprises RNA and cDNA sequences which encode the PB2 tryptophan variant polypeptides of the invention.
The PB2 tryptophan variant RNA sequences of this invention can be rescued into influenza genomes to create influenza master donor virus strains containing the specific mutations desired using the techniques of reverse genetics. Thus, in another aspect the invention comprises recombinant influenza viruses containing such novel PB2 tryptophan variant RNA and polypeptide sequences. These recombinant influenza viruses display attenuated growth characteristics in cultured cells and/or live hosts and are useful as master donor viruses in the preparation of influenza virus reassortants and immunogenic compositions for the prophylactic treatment of humans for influenza infection. To make such recombinant influenza viruses, permissive host cells are infected with a helper virus and transfected with a synthetic RNP complex. The synthetic RNP complex is transcribed in vitro from DNA that encodes the mutated RNA sequence and packaged into ribonucleoprotein (RNP) before transfection. Viral progeny resulting from the transfection includes virus that has incorporated the mutated, transfected RNA sequence into viral particles. Transfectant viruses recovered from the cells that have incorporated the mutated, transfected sequence are then selected from the mixture of transfectant and helper virus, exploiting a phenotypic difference between the two viruses. These transfectant viruses so selected comprise the recombinant influenza viruses of the invention. In such embodiment, the modified tryptophan variant PB2 sequence will contain one or more deletions, replacements or substitutions of the tryptophan amino acid residues giving rise to attenuating phenotypes.
In yet another aspect the invention comprises a method of producing modifications in an influenza genome comprising introducing a recombinant, negative strand RNA template encoding a PB2 tryptophan variant polypeptide into cells infected with a helper virus capable of producing influenza virus RNA segments. One helper virus which can be employed is capable of growth in avian cells but not in mammalian cells. More specifically for example, Madin-Darby bovine kidney (MDBK) or primary chick kidney (PCK) cells can be infected with a host-range mutant of influenza containing the PB2 gene of an avian virus. See Clements, J Clin Microbiol 30:655-662 (1992). Synthetic PB2 RNP is then prepared by in vitro transcription of a cDNA template encoding the mutated, vRNA-sense, PB2 RNA in the presence of purified RNP proteins. The cDNA must encode a PB2 protein which, when rescued into the helper virus, allow it to form plaques in mammalian cells. The resulting RNP is introduced into the infected MDBK or PCK cells, the cells incubated and the medium harvested and used to infect MDCK cells.
In yet another aspect, the invention comprises a reassortant virus including RNA sequences encoding the HA and NA glycoproteins derived from a wide-type epidemic strain of influenza virus, and the remaining RNA sequences derived from the transfectant virus. The wide-type epidemic virus is a circulating strain of influenza virus against which immunity is desired. The transfectant virus is the attenuated master donor, i.e. recombinant influenza virus of the invention which contains attenuating mutations in one or more of the native tryptophan residues in the PB2 sequences of the invention as disclosed herein which can be created and tested for attenuation following the methods described herein. The most reproducible way to generate a suitably attenuated vaccine virus is to retain all six of the internal protein RNA segments (PB1, PB2, PA, NP, M, and NS) of the master donor; however, it may also be possible to have fewer master donor segments in the vaccine virus but still maintain an appropriate level of attenuation, and genetic stability.
In yet another aspect, the invention comprises immunogenic pharmaceutical compositions containing an immunogenically-inducing effective amount of an influenza virus variant in admixture with a pharmaceutically acceptable carrier or solution.
In yet another aspect the invention comprises a method for the prophylactic treatment of a patient comprising administering an immunogenically-inducing effective amount of an immunogenic pharmaceutical composition of the invention to such patient. By xe2x80x9cimmunogenically-inducingxe2x80x9d we mean an amount sufficient for stimulating in a mammal the production of protective antibodies to influenza. Such an amount may stimulate antibody production locally and/or systemically, thereby preventing infection or the disease caused by such infection. Preferably, the patient is a human patient.
In this disclosure, reference is made to the common amino acids using the conventional single-letter symbols.
The modification of tryptophan residues in the influenza virus native PB2 protein or polypeptide (which terms xe2x80x9cproteinxe2x80x9d and xe2x80x9cpolypeptidexe2x80x9d are used interchangeably herein) results in the exhibition of attenuation, and surprisingly, temperature sensitivity in the virus. Such modification encompasses deletion of the native tryptophan residue, or substitution or replacement of the native tryptophan amino acid residue with a non-native amino acid residue. Preferred amino acids for replacement or substitution include phenylalanine, tyrosine and histidine. Especially preferred is phenylalanine. Ten tryptophan amino acid residues were identified in the native influenza A virus A/LA/2/87 PB2 protein. These are located at amino acids 49, 78, 98, 99, 240, 449, 537, 552, 557, and 564, using the conventional numbering counting from the N-terminal MET residue as 1.
Analysis of amino acid sequences of the PB2 proteins from numerous other influenza A strains identified the corresponding tryptophan residues in those strains. Such influenza A strains include A/Memphis/8/88, A/Chile/1/83, A/Kiev/59/79, A/Udorn/307/72, A/NT/60/68, A/Korea/426/68, A/Great Lakes/0389/65, A/Ann Arbor/6/60, A/Leningrad/13/57, A/Singapore/1/57, A/PR/8/34 and A/WSN/33. Their sequences are available from GenBank and viral stock may be available from the American Type Culture Collection, Rockville, Maryland or are otherwise publicly available. Thus, although the A/LA/2/87 strain was used in the examples, any of the foregoing strains could equally have been used. In addition, analyses for tryptophan residues in the PB2 proteins of influenza B and/or C virus could be readily performed in accordance with the teachings of this invention to create PB2 tryptophan variant proteins and live recombinant influenza B and influenza C viruses in an manner analogous to that demonstrated here for influenza A. For example, tryptophan residues corresponding to positions 49, 98, 99, 240, 449, 537 and 552 in influenza A have been found in two influenza B strains, B/AA/1/66 and B/NY/1/93; those at positions 49, 78, 99 and 240 have also been found in the PB2 protein of influenza C virus C/JJ/50. See Yamashita, Virology 171: 458-66 (1989).
Tryptophan residues can be modified following the teachings here to create attenuated, temperature sensitive recombinant influenza viruses. Such attenuated, temperature sensitive recombinant influenza viruses include those containing the PB2 tryptophan variant amino acid sequences, and their encoding RNA or cDNA sequences, which are responsible for the exhibited attenuation and temperature sensitivity.
Accordingly, this invention discloses and describes novel RNA and corresponding cDNA sequences encoding influenza PB2 tryptophan variant proteins. The proteins of this invention comprise variant or modified influenza PB2 sequences in which at least one and up to four of the tryptophan residues of wild-type (i.e., native) influenza PB2 sequences involved in cap-binding are modified by deletion or by replacement or substitution with other amino acids. Phenylalanine is a preferred replacement amino acid. Other preferred amino acids include tyrosine and histidine. The preferred tryptophan amino acid residues for deletion, substitution or replacement are those residues involved in the cap-binding activity of the PB2 protein. In influenza A, those amino acid residues are believed to be the native tryptophan amino acid residues at positions 537, 552, 557 and 564. The words variant, modified and mutant or mutated are used interchangeably herein. The novel RNA and corresponding cDNA sequences encoding influenza PB2 tryptophan variant proteins may also comprise variant or modified influenza PB2 sequences in which the PB2 ts mutations described in detail in Lawson, Virus Res 191:506-10(1992) and in PCT Patent Publication WO 95/08634 published Mar. 30, 1995 are included. Specifically, such influenza A PB2 polypeptide sequences containing mutations at amino acid positions 65, 100, 112, 174, 298, 310, 386, 391, 556, 658, 265, 417 and 512 (xe2x80x9cts amino acidsxe2x80x9d), and their corresponding mutated codons, in combination with the deletion, replacement or substitution of from one to four PB2 tryptophan residues believed to be involved in cap-binding, also comprise the novel RNA, cDNA and polypeptide sequences of the invention. Mutant PB2 polypeptide sequences containing deletions, substitutions or replacements at ts amino acids 65, 112 and 265 and at from one to four tryptophan residues are preferred. Mutant PB2 polypeptide sequences containing deletions, substitutions or replacements at ts amino acids 65, 112 and 265 and tryptophan residues 552, 557 and 564 are most preferred; and substitution or replacement of those ts amino acids and those tryptophan residues is especially preferred.
Such proteins, when incorporated into influenza viruses to create master donor strains of influenza, result in the creation of attenuated and temperature sensitive mutants useful in the preparation of immunogenic compositions and in the prophylactic treatment of influenza.
The influenza PB2 tryptophan variant proteins (i.e., the influenza PB2 proteins containing one or more deleted, replaced or substituted tryptophan amino acid residues and optionally containing one or more ts deletion, replacement or substitution) of this invention can be incorporated into influenza viruses by employing known genetic reassortment or reverse genetic methods. In reverse genetic methods, the native influenza PB2 nucleotide sequence is replaced with a synthetic gene synthesized in vitro from cDNA. The cDNA has at least one of the codons encoding at least one of the native tryptophan amino acid residues believed to be involved in cap-binding either deleted, or replaced or substituted with nucleotides encoding a non-native amino acid residue. Optionally, the cDNA also has at least one of the codons encoding at least one of the native amino acid residues known to be responsible for the ts phenotype, preferably residues 65 and/or 112 of A/Udorn/307/72 and/or 265 of A/AA/6/60(ca), either deleted, or replaced or substituted with nucleotides encoding a non-native amino acid residue. Preferably, the codons should be modified such that reversion to the wild-type amino acid is less likely, by replacing at least two of the nucleotides with non-native nucleotides. Helper virus infected cells are transfected with the synthetic influenza PB2 tryptophan variant RNA sequence. The live virus containing the synthetic influenza PB2 tryptophan variant RNA and amino acid sequence can serve as a master donor virus, which, when combined with the wild-type HA and/or NA gene of epidemic (i.e., currently circulating virulent) influenza strains, will result in the production of reassortant influenza viruses (xe2x80x9c6:2 reassortantsxe2x80x9d) which can be used as immunogenic compositions in the prophylactic treatment of influenza in human. The 6:2 reassortant viruses will thus be composed of six genes derived from the master donor strain containing the synthetic sequence or sequences and the HA and NA genes derived from a currently circulating virulent strain of influenza. The method of preparing a 6:2 influenza reassortant virus comprises infecting a cell with the attenuated master donor strain and with a currently-circulating virulent influenza A virus and selecting the reassortant virus by inhibiting the replication of viruses containing the HA and NA genes of the master donor strain by incubation with an antibody reactive with those proteins. Alternatively, reverse genetics techniques can be used to transfect cells with the HA and NA genes from an epidemic strain. The cells are then infected with the master donor strain and 6:2 reassortants selected by antibody mediated selection as described above.
For example, primary chick kidney (PCK) or MDBK cell monolayers are infected with helper virus at a multiplicity of infection (moi) of 1-10 for 1 hour. RNA encoding one or more of the tryptophan variant PB2 proteins of the invention is transfected into the infected cells using the techniques described in Luytjes, supra, Enami and Palese, supra and Enami, supra optionally as modified in Example 4 below. The transcription reaction contains linearized plasmid, each of the deoxyribonucleotides, T3 RNA polymerase and ribonucleoprotein prepared from virus grown in the allantoic cavities of embryonated eggs according to the methods of Parvin, supra. The mixture is incubated at 37xc2x0 C. for 45 minutes, resulting in the production of RNA transcripts which are concurrently packaged into RNP complexes. The addition of DNase then eliminates the plasmid and the mixture is introduced into the PCK or MDBK cells, which have been infected with the helper virus and treated with DEAE Dextran. Alternatively, the mixture is introduced into the infected cells by electroporation. Cultures are maintained at the appropriate temperature (e.g. 34xc2x0 C.) and are harvested about 16-22 hours later. Cell debris is pelleted and the supernatant containing the virus is plaqued on appropriate mammalian cells, for example MDCK cells. The progeny of the plaqued virus can go through subsequent additional plaque passages and is then amplified in the allantoic cavities of embryonated eggs.
More specifically, a host-range mutant of influenza virus A/LA/2/87 has been described. This helper virus contains the PB2 gene derived from the avian virus, A/Mallard/New York/6750/78, and is able to grow productively in avian cells such as PCK cells, but cannot form plaques in mammalian cells such as MDCK. See Clements, JClin Microbiol 30:655-62 (1992). Replacement of the Mallard PB2 gene in the helper virus with a transfected, mammalian PB2 sequence allows the virus to plaque in MDCK cells. See Subbarao, J Virol 67:7223-28 (1993). In this way specific alterations in the nucleotide sequence of the PB2 gene can be introduced, by transfecting synthetic RNAs derived from cDNAs of the mammalian PB2 sequence bearing site-directed mutations. The recombinant variant influenza virus so produced will exhibit temperature sensitivity, thereby enabling it to be employed as the master donor strain in the construction of live, attenuated immunogenic compositions for prophylactic administration in humans.
Standard methods may be employed for propagating the recombinant influenza viruses of the invention. Viral stocks can be plaque-purified in primary or established cell cultures, for example, primary bovine or chick kidney cells or MDCK cells. Plaque-purified virus can be further propagated in such cell lines. The cells are cultured typically on plastic tissue culture plates and virus is typically inoculated at a moi of 0.001 to 0.1 and incubated for 2-3 days. Virus stock can alternatively be inoculated into the allantoic cavity of 10-12 day embryonated chicken eggs and incubated for 2-3 days at 33-37xc2x0 C.
Testing for attenuation of the recombinant influenza viruses of the invention can be accomplished employing well established in vitro and in vivo assays. In the in vitro assay, the recombinant virus is tested for the presence of the temperature sensitive phenotype, as described in Example 6 below. Ability to replicate in the respiratory tract of mice can be determined as described in Example 7 below. In vivo reactogenicity of the recombinant influenza viruses can be determined as described in Example 8 below. Phenotypic stability of the recombinant influenza viruses can be determined as described in Example 9 below.
Such recombinant modified, variant influenza viruses can also be used in genetic complementation analysis, to map ts lesions of other viruses, and in the functional analysis of the role of PB2 in the virus life cycle.
The modified PB2 proteins of the invention can be expressed recombinantly in different types of cells using the appropriate expression control systems, as is well known in the art, to test protein functionality. The construction of suitable vectors containing the nucleic acids sequences of the invention is likewise well known in the art, as are hybridization assays in which such sequences may be employed. See for example, U.S. Pat. Nos. 4,356,270 issued to Itakura, U.S. Pat. No. 4,431,739 issued to Riggs and U.S. Pat. No. 4,440,859 issued to Rutter. Other exemplary host cells, promoters, selectable markers and techniques are also disclosed in U.S. Pat. No. 5,122,469 issued to Mather, U.S. Pat. No. 4,399,216 and U.S. Pat. No. 4,634,665 issued to Axel, U.S. Pat. No. 4,713,339 issued to Levinson, U.S. Pat. No. 4,656,134 issued to Ringold, U.S. Pat. No. 4,822,736 issued to Kellems and U.S. Pat. No. 4,874,702 issued to Fiers.
The construction of suitable vectors containing the nucleic acid sequences of the invention is accomplished using conventional ligation and restriction techniques now well known in the art. Site specific cleavage is performed by treatment with suitable restriction enzyme(s) under standard conditions, the particulars of which are typically specified by the restriction enzyme manufacturer. Polyacrylamide gel or agarose gel electrophoresis may be performed to size separate the cleaved fragments using standard techniques. Synthetic oligonucleotides can be made using for example, the diethyphosphoamidite method known in the art. Ligations can be performed using T4 DNA ligase under standard conditions and temperatures, and correct ligations confirmed by transforming E. coli with the ligation mixture. Successful transformants are selected by ampicillin, tetracycline or other antibiotic resistance or using other markers as are known in the art.
Such recombinant techniques are fully explained in the literature. See, e.g., Sambrook, Molecular Cloning: A Laboratory Manual, 2d ed. (1989); DNA Cloning, Vol. I and II, D. N. Glover, ed., 1985; Oligonucleotide Synthesis, M. J. Gait, ed., 1984; Nucleic Acid Hybridization, B. D. Hames, ed., 1984; Transcription and Translation, B. D. Hames, ed., 1984; Animal Cell Culture, R. I. Freshney, ed., 1986; B. Perbal, A Practical Guide to Molecular Cloning (1984); Gene Transfer Vectorsfor Mammalian Cells, J. H. Miller, ed., 1987, Cold Spring Harbor Laboratory; Scopes, Protein Purification: Principles and Practice, 2d ed, Springer-Verlag, New York, 1986 and Handbook of Experimental Immunology, Vols I-IV, D. M. Weired, ed., 1986. All such publications mentioned herein are incorporated by reference for the substance of what they disclose.
The live recombinant influenza virus variants of the invention may be employed in immunogenic compositions for preventing infection by an influenza virus or the disease state brought about by such infection. To make such immunogenic compositions, cultured cells are co-infected with the live recombinant influenza variant (i.e., the master donor) and an epidemic wild-type strain. Reassortant viruses are harvested and tested for the presence of the mutation in the native tryptophan residue. Reassortants containing the wild-type HA and/or NA proteins can be selected by exposure to antisera against the surface epitopes encoded by the HA and/or NA proteins from the donor virus. Resultant viral progeny containing the mutated sequences of the invention and the HA and/or NA sequences from the wild-type epidemic influenza strains are used in the preparation of immunogenic compositions. Such immunogenic compositions comprise an immunogenically-inducing effective amount of a recombinant influenza virus variant of the present invention in admixture with a pharmaceutically acceptable carrier or solution. An exemplary pharmaceutically acceptable carrier is saline solution. The composition can be systemically administered, preferably subcutaneously or intramuscularly, in the form of an acceptable subcutaneous or intramuscular solution. More preferably, the composition can be administered intranasally, either by drops, large particle aerosol (greater than 10 microns), or spray into the upper respiratory tract. The preparation of such solutions, having due regard to pH, isotonicity, stability and the like is within the skill in the art. The dosage regimen will be determined by the attending physician considering various factors known to modify the action of drugs such as for example, age, physical condition, body weight, sex, diet, time of administration and other clinical factors. Exemplary dosages range from about 1 to about 1000 HID50 (human infectious dose) of the virus.
In practicing the method of prophylactic treatment of this invention, an immunologically-inducing effective amount of an immunogenic composition of the invention is administered to a human patient in need of prophylactic treatment. An immunologically inducing effective amount of a composition of this invention is contemplated to be in the range of about 1-1000 HID50, i.e., about 105-108 pfu (plaque forming units) per dose administered. The number of doses administered may vary, depending on the above-mentioned factors. The route of delivery will preferably be via nasal administration into the upper respiratory tract of the patient.